Field of the Invention
The present invention relates to the field of molecular biology. More specifically, the present invention relates to methods of performing Polymerase Chain Reactions (PCR) and to improved polymerases for performing PCR.
Description of Related Art
DNA polymerases are enzymes capable of catalyzing the replication of DNA and have become indispensable research tools in biotechnology. In particular, the thermostable DNA polymerase from Thermus aquaticus, Tag, is commonly used in PCR reactions. Cyclic polymerase-mediated reactions, such as PCR, have numerous applications in the fields of basic research, medical diagnostics, and forensics.
In a standard protocol, PCR is based on three repeated steps: denaturation of a DNA template, annealing of primers to the denatured DNA template, and extension of the primers with a polymerase to synthesize nucleic acids complementary to the template. The conditions under which these steps are performed are well established in the art.
One important characteristic of the thermostable DNA polymerase used in a PCR reaction is its polymerization rate. A fast polymerase is desirable because it allows for shorter extension cycle times, resulting in production of amplification products in a shorter period of time than a slower polymerase. An amplification run thus may be shortened, allowing more efficient use of researchers' time, higher throughput on cost-limiting PCR equipment, and rapid medical diagnostic applications. Polymerization rate, or the number of nucleotides incorporated per unit time (under specified reaction conditions including temperature, pH, ionic strength, etc.), is influenced by a number of parameters including binding affinity for substrates (dNTPs, primed DNA) and catalytic efficiency (rate of nucleotidyl transfer, pyrophosphate release, and translocation steps). Processivity, or the number of nucleotides incorporated per binding event, is largely influenced by the affinity of polymerase for template. Polymerases with increased polymerization rate or increased processivity provide several benefits to PCR, including the ability to use faster cycling times.
Attempts to increase processivity of DNA polymerase used in PCR have included the use of fusion proteins containing DNA polymerase activity. For example, DNA polymerases have been fused to a domain for binding known processivity factors, such as thioredoxin or an Archaeal proliferating cell nuclear antigen. DNA polymerases have also been fused to multiple helix-hairpin-helix motifs identified in DNA topoisomerase V and to a sequence non-specific dsDNA binding protein.
Another strategy to improve PCR performance under fast cycling conditions is to mutate the polymerase. In this case, other characteristics of the enzyme may be reduced or eliminated, such as the 5′-3′ exonuclease activity (see, for example, U.S. Pat. No. 5,474,920).
Another desirable characteristic of a DNA polymerase to be used in PCR reactions is its ability to work in complex or “dirty” environments. The ability of a DNA polymerase to polymerize in these kinds of samples significantly increases the applicability of the polymerase. Complex biological samples such as blood, cell lysates, plants and plant extracts, environmental samples, etc. have many components that can inhibit DNA polymerases used in PCR reactions. These components include hemoglobin, immunoglobulin G, lactoferrin, and perhaps protease activity in blood. Soil sample components that interfere with PCR reactions include humic acid, fulvic acid, plant polysaccarides, and metal ions. Although various procedures have been developed to pre-treat samples before attempting PCR reactions, these steps are generally time-consuming, labor-intensive, and might not achieve the purification required for the subsequent PCR. In addition, precious nucleic acid can be lost from the sample before the PCR reaction step.
Strategies to improve PCR reactions using complex samples without pre-treatment include the addition of substances to the PCR reaction, which can reduce the effect of PCR inhibitors found in the sample. For example, the addition of bovine serum albumin or the addition of single-stranded DNA binding T4 gene 32 protein to a PCR reaction mixture is known to enhance the amplification capacities of some DNA polymerases.
As with strategies that have been used to develop DNA polymerases with increased processivity, one strategy to improve the ability of DNA polymerases to perform PCR reactions in complex samples involves mutating the DNA polymerase itself. For example, it is known that N-terminal deletions of Taq DNA polymerase and/or mutations of certain amino acids can confer enhanced resistance to various inhibitors of PCR reactions. However, very few mutant DNA polymerases suitable for use in “dirty” PCR reactions have been reported.